(a) Field of the Invention
The invention relates to new paclitaxel derivatives and the use of such paclitaxel derivatives in the synthesis of immunoconjugates, all those compounds aimed for cancer therapy.
(b) Description of Prior Art
Paclitaxel is a natural product extracted from the bark of the Pacific yew (Taxus brevifolia). It was thereafter found in other members of the Taxacae family including the yew of Canada (Taxus canadensis) found in Gaspesia, eastern Canada and Taxus baccata found in Europe whose needles contain paclitaxel and analogs and hence provide a source of paclitaxel and derivatives. The crude extract was tested for the first time during the 60s and its active principle was isolated in 1971 by Wani et al. who at the same time identified its chemical structure. Paclitaxel is a microtubule blocker, but unlike other drugs inhibiting the mitosis by interaction with microtubules such as colchicin, vincristin and podophyllotoxin, paclitaxel does not prevent tubulin assembly. It rather accelerates the tubulin polymerization and stabilizes the assembled microtubules. The drug acts in a unique way that consists in binding to microtubules, preventing their depolymerization under conditions where usually depolymerization occurred (dilution, calcium, cold and microtubules disrupting drugs). Paclitaxel blocks the cell cycle at prophase, which results in an accumulation of cells in G2+M. Because of its unique structure and mechanism of action, paclitaxel is currently used in the treatment of ovarian, breast and non-small cell lung cancers.
Poor solubility of paclitaxel constitutes an important limitation to its administration to cancer patients. To increase paclitaxel availability, total and partial synthesis has been reported. The improvement of paclitaxel solubility was obtained by adjunction of solubilizing functions such as carbonyl or sulfonyl groups with good results. Some of the synthesized products were more active than paclitaxel, many others had a biological activity equivalent or slightly inferior to that of paclitaxel while being far more soluble in water (KINGSTON, D. G., Pharmacol. Ther. (England), 52(1) p1-34, 1991).
Paclitaxel has three hydroxyl groups at carbon 1, 7 and 2xe2x80x2 susceptible of undergoing an acylation. Their reactivity varies according to the following order: 2xe2x80x2 greater than 7 greater than  greater than  greater than 1 (MATHEW, A. E., et al., J. Med. Chem., 35, 145-151, 1992). Acylation on 2xe2x80x2C is the best way of paclitaxel modification because of its great reactivity, and because even if 2xe2x80x2 acylpaclitaxels loose their property of promoting the microtubules polymerization in vitro, they are hydrolyzed in the cell and revert to paclitaxel and keep their cytotoxic activity (KINGSTON, D. G., et al., J. Nat. Prod., 1-13, 1990; and MELLADO, W., et al., Biochem. Biophys. Res. Commun., 105, 1082-1089, 1984).
Accordingly, to increase solubility and in order to add a functional group that allows the coupling of paclitaxel to a protein carrier, several derivatives have been synthesized by modification of the 2xe2x80x2 hydroxyl group.
Chemotherapeutic agents currently used for antitumor therapy are selected for their toxicity towards rapidly proliferating cells. Most of them cause undesirable systemic effects such as cardiac or renal toxicity, marrow aplasia, alopecia, nausea and vomiting. During the last few years, many authors have tried to eliminate these side effects by increasing the availability of the drug to the tumour site. In fact, delivery of the drug can frequently be as important as the activity of the drug itself in providing an effective treatment.
The targeting of drugs to a tumour by antibodies to surface antigens may have considerable implications by increasing the therapeutic index.
Several coupling methods permit the linkage of a cytotoxic drug to a protein that targets a specific antigen expressed by a specific type of cell such as cancer cells. The inventor has described a coupling method of an anti-tumor to an antibody using glutaraldehyde preactivated anti-tumor agent in U.S. Pat. No. 5,208,323, the content of which is hereby incorporated by reference. In that method, an amino group of anticancer drugs such as anthracyclines or daunorubicine is activated with glutaraldehyde. The latter is then bound to specific antibodies for cancer cells. The inventor has already described some soluble derivatives of paclitaxel, among which taxamine has a free amine group that may be activated by glutaraldehyde for coupling.
The principle of this targeting is that once inside the cell the imine bond is lysed by lysosomal enzymes. These may be active on the product if steric hindrance does not prevent its action.
If one attach an active molecule to a longer atom, the steric hindrance will be decreased. Such molecules have been used and commercialized for coupling biotin to macromolecule. Side arms made of C6 to C8 have been used successfully.
It would be highly desirable to be provided with new active paclitaxel derivatives. It would thus be highly desirable to be provided with a drug targeting method to administer new derivatives of paclitaxel for drug targeting cancer treatment.
It would also be highly desirable to be provided with new immunoconjugates to specifically target cancer cells.
One aim of the present invention is to provide a new active paclitaxel derivative modified at the C-2xe2x80x2 position.
Another aim of the present invention is to provide new paclitaxel derivatives, which show cytotoxic activity similar to paclitaxel alone.
Another aim of the present invention is to provide a pharmaceutical composition comprising a derivative as defined above with a pharmaceutically acceptable carrier.
Another aim of the present invention is to provide an immunoconjugate comprising a new paclitaxel derivative conjugated to a carrier molecule.
Another aim of the present invention is to provide a method to conjugate a paclitaxel derivative as defined above to a carrier molecule.
Another aim of the present invention is to provide a method for in vivo treatment or prophylaxis of cancer.
In accordance with the present invention there is provided a new paclitaxel derivative or a salt thereof having the following Formula I: 
wherein R is a xe2x80x94COxe2x80x94(CH2)4-8xe2x80x94COX, in which X is selected from the group consisting of a hydroxyl, a 1,2-diaminoethenyl, a 1,3-diaminopropyl, a 1,4-diaminobutyl, a 1,5-diaminopentyl, a 1,6-diaminohexyl, and a polar amino acid residue.
Preferably, the polar amino acid residue is selected from the group of residues consisting of arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, phenylalanine, serine, threonine and tyrosire.
In accordance with the present invention, there is also provided a method for the in vivo treatment or prophylaxis of cancer comprising the step of administering a therapeutically effective amount of a new paclitaxel derivative as defined above to a patient in need of such a treatment.
In accordance with the present invention, there is also provided an immunoconjugate having the following formula II:
Rxe2x95x90(CH)xe2x80x94(CH2)mxe2x80x94(R1)nxe2x80x94(CH2)pxe2x80x94(CH)xe2x95x90Mxe2x80x83xe2x80x83II
wherein R1 is 
and wherein
m and p are an integer varying from 0 to 3, with one of
m and p being 0;
n is an integer varying from 1 to 3;
M is selected from the group consisting of a peptide residue and a protein residue linked to the carbon atom via an amino residue of lysine present therein; and
R represents a new derivative of paclitaxel as defined above attached at its C-2xe2x80x2 position.
In a preferred embodiment of the immunoconjugate of formula II, m is 3 when p is 0 or m is 0 when p is 3.
The method of the present invention as described above can be easily produced and is devoid of significant polymerization.
In accordance with the present invention, there is also provided a method for the in vivo treatment or prophylaxis of cancer comprising the step of administering a therapeutically effective amount of an immunoconjugate comprising a new paclitaxel derivative as defined above to a patient in need of such a treatment.
In accordance with the present invention, there is further provided the use of a derivative as defined previously for the preparation of a medicament for treating cancer.
In accordance with the present invention, there is further provided a method of preparing compounds of Formula II:
Rxe2x95x90(CH)xe2x80x94(CH2)mxe2x80x94(R1)nxe2x80x94(CH2)pxe2x80x94(CH)xe2x95x90Mxe2x80x83xe2x80x83II
wherein R1 is 
and wherein
m and p are an integer varying from 0 to 3, with one of m and p being 0;
n is an integer varying from 1 to 3;
M is selected from the group consisting of a peptide residue and a protein residue linked to the carbon atom via an amino residue of Lysine present therein; and
R represents a new derivative of paclitaxel as defined above attached at its C-2xe2x80x2 position. The method comprises the steps of:
a) reacting the new derivative of paclitaxel with glutaraldehyde for obtaining a new paclitaxel derivative glutaraldehyde complex; and
b) reacting the new paclitaxel derivative glutaraldehyde complex of step a) with a protein or a peptide for obtaining an immunoconjugate.
In order to couple paclitaxel to a carrier molecule, it was then appropriate to synthesize new molecules with long chain (C6-C10) dicarboxylic acid side arms extended by either ethylenediamine, propanediamine, butanediamine, pentanediamine or hexanediamine groups. The final product may then be activated and coupled.
In a preferred embodiment of the invention, M is a protein selected from the group of BCM43 monoclonal antibody, BCM43 humanized antibody, BCM43 chimerized antibody, the (Fab), (Fab)2 or scFv moiety of the BCM43 antibody, BCM17 monoclonal antibody, BCM17 humanized antibody, BCM17 chimerized antibody, the (Fab), (Fab)2 or scFv moiety of the BCM17 antibody, anticarcinoembryonic monoclonal antibody and antialphafetoprotein monoclonal antibody. BCM43 is a monoclonal antibody recognizing an epithelial mucin expressed by ovarian and breast cancer cells. BCM17 (monoclonal anti-P7) is a murine monoclonal antibody recognized a P7 protein, which is overexpressed in resistant ovarian cancer. The hybridoma producing the BCM43 monoclonal antibody was deposited in the International Depositary Authority of Canada in accordance with the Budapest treaty on Sep. 28, 1999, and was given accession number IDAC 280999-1.
In accordance with the present invention, there is provided a method for treating cancer, which comprises administering to a patient a therapeutic dosage of a compound of Formula II as defined previously.
The compound of the present invention may be formulated in a pharmaceutical composition. Such composition, or the compound itself, is preferably used for the preparation of a medicament for treating cancer.